Disease resistance is an important objective of the genetic engineering of crop plants. Numerous fungi are serious pests of common agricultural crops. The maize plant is susceptible to a variety of pathogenic fungi that reduce yield and quality of the crop all over the world. In the United States alone annual losses in the Corn Belt range from about 7% to about 17%. One method of controlling diseases has been to apply antifungal organic or semiorganic chemicals to crops. This method has numerous, art-recognized problems. A more recent method of control of microorganism pests has been the use of biological control organisms which are typically natural competitors or inhibitors of the troublesome microorganisms. However, it is difficult to apply biological control organisms to large areas, and even more difficult to cause those living organisms to remain in the treated area for an extended period. Still more recently, techniques in recombinant DNA have provided the opportunity to insert into plant cells cloned genes which express antifungal compounds. This technology has given rise to additional concerns about eventual microbial resistance to well-known, naturally occurring antifungals, particularly in the face of heavy selection pressure, which may occur in some areas. Thus, a continuing effort is underway to express naturally occurring antifungal compounds in plant cells directly by translation of a single structural gene.
However, there is a limited pool of naturally occurring peptides and other compounds with which molecular biologists can work. Attention is now focused on the rational design of entirely new peptides which can function effectively in plant cell expression systems and in other uses where antifungal peptides can be used.
In addition, there are other aspects of plant cell expression systems which make the design of new antifungal peptides desirable. Crop plants have more important things to do than fight disease. They are sources of sugars, starches, proteins, oils, fibers, and other raw materials. Genetic engineers would also like to modify, and often to enhance, the production of those natural plant products.. Unfortunately, plant cells can only produce large quantities of a few cellular components at a time. If they are producing high levels of storage proteins, it is difficult for them to also produce high levels of antifungal compounds. Thus, genetic engineers face a quandary in designing advanced plant systems which require high-level expression of multiple genes. The creation of entirely new antifungal peptides offers the molecular designer the opportunity to select structures which enhance the plant's content of various important or limiting amino acids while also providing antifungal activity. One example of this is the copending application of Rao and Beach, "High Lysine Derivatives of Alpha-Hordothionin", No. 08/003,885, filed Jan. 13, 1993, now abandoned. Even so, there continues to exist a need for still more compounds which can be evaluated and used in various plant and non-plant antifungal applications.
The principle of amphipathy has been used in the past to design biologically active proteins. In 1981 De Grado et al., J. Am. Chem. Soc. 103:679-681 showed that the completely synthetic analog of melittin was biologically active even though it had no homology to the natural peptide. Fink et al., Int. J. Pep. Prot. Res. 33:412-421 (1989) and Boman et al. FEBS Lett. 1: 103-106 (1989) have demonstrated antibacterial activity of synthetic cecropin-like model peptides and cecropin-melittin hybrid compounds. Lee et al., Biochem. Biophys. Acta 862: 211-219 (1986) and Agawa et al., J. Biol. Chem. 266:20218-20222 (1991) have shown a relationship between antifungal activity and amphiphilic properties of basic model peptides. More recently, Moser, Protein Eng. 5: 323-331 (1992) has reported on the design., synthesis and structure of an amphipathic peptide with pH-inducible hemolytic activity. Taylor et al., Molec.Pharm. 22:657-666 (1982) have synthesized analogs of beta-endorphin possessing complete biological activity. Frohlich and Wells, Int. J. Pep. Prot. Res. 37: 2-6 (1991) have suggested the idea of peptide amphipathy in the design of mechanism-based insecticides.